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Am J Physiol Gastrointest Liver Physiol 292: G154-G164, 2007. First published September 28, 2006; doi:10.1152/ajpgi.00173.2006
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MUCOSAL BIOLOGY

M2 and M3 muscarinic receptors are involved in enteric nerve-mediated contraction of the mouse ileum: findings obtained with muscarinic-receptor knockout mouse

Tadayoshi Takeuchi,1 Keisuke Tanaka,1 Hidemitsu Nakajima,1 Minoru Matsui,2 and Yasu-Taka Azuma1

1Department of Veterinary Pharmacology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai Osaka, Japan; and 2Division of Neuronal Network, Department of Basic Medical Sciences, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Submitted 26 April 2006 ; accepted in final form 11 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The involvement of muscarinic receptors in neurogenic responses of the ileum was studied in wild-type and muscarinic-receptor (M-receptor) knockout (KO) mice. Electrical field stimulation to the wild-type mouse ileum induced a biphasic response, a phasic and sustained contraction that was abolished by tetrodotoxin. The sustained contraction was prolonged for an extended period after the termination of electrical field stimulation. The phasic contraction was completely inhibited by atropine. In contrast, the sustained contraction was enhanced by atropine. Ileal strips prepared from M2-receptor KO mice exhibited a phasic contraction similar to that seen in wild-type mice and a sustained contraction that was larger than that in wild-type mice. In M3-receptor KO mice, the phasic contraction was smaller than that observed in wild-type mice. Acetylcholine exogenously administrated induced concentration-dependent contractions in strips isolated from wild-type, M2- and M3-receptor KO mice. However, contractions in M3-receptor KO mice shifted to the right. The sustained contraction was inhibited by capsaicin and neurokinin NK2 receptor antagonist, suggesting that it is mediated by substance P (SP). SP-induced contraction of M2-receptor KO mice did not differ from that of wild-type mice. SP immunoreactivity was located in enteric neurons, colocalized with M2 receptor immunoreactivity. These results suggest that atropine-sensitive phasic contraction is mainly mediated via the M3 receptor, and SP-mediated sustained contraction is negatively regulated by the M2 receptor at a presynaptic level.

M2- and M3-receptor knockout mouse; neurogenic response; acetylcholine-mediated phasic contraction; substance P-mediated tonic contraction

ACETYLCHOLINE (ACh) is released from cholinergic nervous terminals in the myenteric plexus, resulting in the contraction of intestinal smooth muscle through activation of muscarinic (M) receptors (9). Five subtypes of M receptors have so far been identified (39). It is well known that M2 and M3 receptors are present on the smooth muscle cells of gastrointestinal tracts and are expressed at ratios of 70–80% and 20–30%, respectively (6). A role of both M receptors in smooth muscle contraction was indicated by the examination of the effects of M-receptor subtype-specific antagonists on the contraction induced by exogenous M-receptor agonists (7). In the guinea pig and canine ileum, contractions induced by various M-receptor agonists (carbachol, muscarine, and methacholine) were most strongly inhibited by the M3-receptor antagonist 4-diphenylacetoxy-N-methyl-piperidine methiodide (12, 27). In contrast, activation of the M2 receptor in rat and guinea pig ileum was reported to inhibit isoproterenol-induced relaxation, suggesting that M2 receptors were involved indirectly in smooth muscle contraction by suppressing the relaxant response (10, 33). These results suggest that the M3 receptor is an important subtype in the contraction of intestinal smooth muscle. Recently, however, the necessity for reevaluating the roles of the M-receptor subtype in contraction was raised because it became apparent that the affinity of muscarinic antagonists was low among the various M-receptor subtypes (5, 40).

Mutant mice lacking either the M2- or M3- or both receptor subtypes have been generated by gene-targeting techniques (20). In ileal samples prepared from M2-receptor knockout (KO) mice, muscarinic agonist added exogenously induced contractions to an extent similar to those seen in the wild-type mice (18, 36). In contrast, carbachol produced a smaller contraction in the ileum from M3-receptor KO mice than seen in the ileum of wild-type mice and did not induce contractions in the ileum isolated from M2- and M3-receptor double-KO mice (18, 19). The results obtained from M-receptor KO mice support the hypothesis that, in the mouse ileum, muscarinic agonists produce contraction(s) mainly through the activation of the M3 receptor and partially through activation of the M2 receptor. However, these results were obtained by studying contractile responses to exogenously added muscarinic agonists. Recently, it has been reported that contractile responses mediated by ACh released from the enteric cholinergic neurons are modulated by several types of intestinal constituents (38). Therefore, it seems pertinent to compare the contractions induced by excitation of enteric neurons in the wild-type mice with those in the M-receptor KO mice.

It was reported that noncholinergic excitatory substances exist in the gastrointestinal tract (16). Tachykinins and ATP were reported to be candidates in the production of muscarinic antagonist-resistant contraction. The results of a charcoal transit test to monitor gastrointestinal activity in M3-receptor KO mice showed that the lack of M3 receptors had no significant effect on gastrointestinal motility in vivo (41), suggesting an essential role of noncholinergic substances in gastrointestinal motility. Although it was previously reported that noncholinergic components of contraction in response to electrical stimulation were stronger in M2- and M3-receptor double-KO mice than in wild-type mice (18), the mechanism remains to be clarified. In the present study, we compared the neurogenic contractile responses induced by electrical field stimulation (EFS) between wild-type and M-receptor KO mice to clarify the role of M-receptor subtype in enteric nerve-mediated contraction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The generation of homozygous M2-, M3-, and M4-receptor KO mice and M2- and M3-receptor double-KO mice has been described previously (8, 15, 18, 19). KO mice were backcrossed with C57BL/6JJcl mice (CLEA, Tokyo, Japan) for 8–14 generations. Age-matched wild-type C57BL/6 mice were also included. A mouse genotyping was carried out by PCR analysis of mouse tail DNA. Adult (2–6 mo old) mice were used in this study. Animal maintenance and experimental procedures were performed under the approval of the guidelines of the ethics committees of Osaka Prefecture University and the Institute of Medical Science (University of Tokyo, Tokyo, Japan). The mice were lightly anesthetized with ether and then stunned by a blow to the head and bled via the carotid arteries. Segments of the ileum were removed and placed in Tyrode solution consisting of (in mM) 137 NaCl, 2.7 KCl, 1.8 CaCl2, 1.1 MgCl2, 0.42 NaH2PO4, 11.9 NaHCO3, and 5.6 glucose. The contents of the excised segments were gently flushed with Tyrode solution. Segments, 2.0 cm in length, were excised from the central part of the ileum.

Recording of responses of longitudinal ileal muscle to EFS. As described in a previous report (25), segments of the ileum were suspended in an organ bath containing 4 ml of Tyrode solution maintained at 37°C and bubbled with a mixture of 95% O2-5% CO2. One end of each segment was attached to an isotonic transducer (TD-112A; Nihonkohden, Tokyo, Japan), and the other end was mounted on an anodal electrode placed at the bottom of the bath. After an equilibration period of 30 min, responses along the longitudinal axes to EFS with trains of 10–100 pulses of 0.5-ms width, 30-V intensity, and 1- to 10-Hz frequency were recorded isotonically with a 10-min interval between tests. When noncholinergic responses were recorded, atropine (1 µM) was present to block the cholinergic response. Drugs were added to the bathing fluid only when responses to EFS became reproducible. The segment was subjected to a resting load of 0.5 g along the longitudinal axis. The extent of contraction was expressed as the percentage of that induced by 60 mM K+ (hypertonic solution), which was carried out at the end of the experiment.

Concentration-response curves for ACh and SP. To measure concentration-response curves for ACh and substance P (SP), increasing concentrations of ACh and SP were applied by a "single-dose" protocol. Each concentration was applied for 1–2 min, followed by washing three or four times with fresh Tyrode solution. The time interval between successive ACh and SP applications was 10 min. ACh- and SP-induced contractions were expressed as the percentage of the contraction induced by 60 mM K+ in the same preparation.

Antibody. Rat monoclonal antisera against M2-muscarinic receptor (MAB367) were purchased from Chemicon International (Temecula, CA). Guinea pig polyclonal antisera against SP (T-5019) and rabbit polyclonal antisera against synaptophysin (08-1130) were purchased from Peninsula Laboratories (San Carlos, CA) and Zymed Laboratories (San Francisco, CA), respectively.

Immunohistochemical study. An immunohistochemical study was carried out by the method described previously (31). Briefly, the intestine was isolated after the wild-type mice were deeply anesthetized with pentobarbital sodium (50 mg/kg ip), and the intestine was fixed by transcardiac perfusion. The intestine was dissected, postfixed with 4% (wt/vol) paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 h, dehydrated with 30% (wt/vol) sucrose solution, and then frozen with OCT compound (Tissue-Tek; Sakura Finetechnology, Tokyo, Japan). Sagittal sections (12 µm) were cut on a cryostat and thaw-mounted on aminosilant-coated slides. For whole-mount preparations, short segments of the intestine were inflated and the mucosa was removed with a small razor; the remaining strips (5 x 5 mm) were pinned to the silicon rubber. The tissues were fixed for 2 h at room temperature with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Both preparations were washed three times with PBS and then placed in PBS containing 0.5% Triton X-100, 1% BSA, and 10% normal goat serum for 1 h at room temperature to avoid nonspecific staining. The preparations were then incubated with antiM2 receptor (1:200), anti-SP (1:10,000), or anti-synaptophysin antibodies (1:200) in PBS at 4°C for 24 h. Immunoreactivity of M2 receptor and SP antibodies was detected by either Alexa fluor 488-conjugated anti-rat IgG (Molecular Probes, Eugene, OR) or indocarbocyanine-conjugated anti-guinea pig IgG (Jackson Immuno Research Laboratories, West Grove, PA) secondary antibodies, respectively. Immunoreactivity of synaptophysin antibody was detected by either Alexa fluor 488-conjugated anti-rabbit IgG (Molecular Probes) or indocarbocyanine-conjugated anti-rabbit IgG (Jackson Immuno Research Laboratories) secondary antibodies. Confocal images were obtained under a laser scanning microscope (MRC-1024; Bio-Rad, Hertfordshire, UK).

Statistical analysis. Only one preparation was made from each animal. Thus n in the results indicates the number of animals. All values are expressed as means ± SE. Statistical analysis of the data was carried out with paired t-test and evaluated by ANOVA and thereafter assessed by either Student's t-test or Welch test (if significant differences were indicated by ANOVA). To estimate the effect of capsaicin, we used a multiple comparison procedure of Dunnett. P values <0.05 were regarded as significantly different.

Drugs. Atropine sulfate, acetylcholine chloride, L-arginine, capsaicin, papaverine, and tetrodotoxin were purchased from Wako Pure Chemical (Osaka, Japan). NG-nitro-L-arginine (L-nitroarginine), {alpha}-chymotrypsin, and MEN-10376 were purchased from Sigma (St. Louis, MO). SP and spantide were purchased from Peptide Institute (Osaka, Japan). Drugs were added to the organ bath in a volume of <1.0% of the bathing solution. These volumes of the vehicle of the drugs (redistilled water) did not affect the spontaneous contractile activity or muscle tone.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In ileum of wild-type mouse, EFS at frequencies ranging from 1 to 10 Hz induced a rapid phasic contraction followed by a sustained contraction (Fig. 1A). The sustained contraction persisted even after the cessation of EFS. Both contractions increased in a frequency-dependent manner (Fig. 1). The muscle strips prepared from M2-receptor KO mice produced contractile responses similar to those of wild-type mice. In the M3-receptor KO mice, however, contraction followed a transient small relaxation and a few minutes of lag time after the start of EFS (Fig. 2B). In addition, the phasic contractions of the M3-receptor KO mouse ileum were significantly lower than those of wild-type and M2-receptor KO mice (Fig. 1B). By contrast, a sustained contraction induced by EFS at 10 Hz was larger in M2-receptor KO mice than in wild-type mice (Fig. 1C). Tetrodotoxin (1 µM) completely inhibited the contractions induced by EFS at all frequencies and in all preparations.


Figure 1
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  		Fig. 1. Frequency-dependent responses of wild-type mouse ileum to electrical field stimulation (EFS). A: representative trace of EFS-induced contraction. The preparations prepared from wild-type mouse ileum were stimulated by EFS at 1, 3, and 10 Hz for 10 s. The highest portions of contraction within and after EFS were measured as the phasic and sustained contractions, respectively. After normal spontaneous movements were recorded, the chart was run at a fast speed immediately before the stimulation to make the contraction clear. Bold horizontal lines indicate the duration of EFS for 10 s. B: summary of EFS-induced phasic contraction in wild-type and muscarinic M2- and M3-receptor knockout (KO) mice. Phasic contractions at 1, 3, and 10 Hz are expressed as a percentage of 60 mM K+-induced contraction. Note that phasic contractions in M3-receptor KO mice were lower than those of the other 2 types of mice. Values are means ± SE for 4–8 experiments. *Significantly different from values for wild-type mice, P < 0.05. C: summary of EFS-induced sustained contraction in wild-type and M2- and M3-receptor KO mice. Sustained contractions at 1, 3, and 10 Hz are expressed as a percentage of 60 mM K+-induced contraction. Note that sustained contractions at 10 Hz in M2-receptor KO mice were higher than those of the other 2 types of mice. Values are means ± SE for 4–8 experiments. *Significantly different from values for wild-type mice, P < 0.05.

 

Figure 2
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  		Fig. 2. Effects of atropine and/or L-nitroarginine on EFS-induced contractions in wild-type (A) and M3-receptor KO (B) mice. Ileal preparations from wild-type and M3-receptor KO mice were stimulated by EFS at 10 Hz for 10 s. Note that EFS in M3-receptor KO mice induced relaxation despite the absence of atropine. Horizontal lines indicate the presence of the indicated drugs. After normal spontaneous movements were recorded, the chart was run at a fast speed immediately before the stimulation to make the contraction clear. Bold horizontal lines indicate the duration of EFS for 10 s.

 
In the presence of atropine (1 µM), EFS at 10 Hz induced a transient relaxation (15.0 ± 4.6% of 30 µM papaverine-induced relaxation; n = 6) in wild-type mice (Fig. 2A). This relaxation was completely inhibited by L-nitroarginine, a nitric oxide synthase (NOS) inhibitor (30 µM; n = 6) (Fig. 2A). Because this result indicated a release of NO, a substance known to influence relaxation, during EFS, subsequent experiments were carried out in the presence of L-nitroarginine to eliminate the influence of NO on contractile responses. In the presence of 30 µM L-nitroarginine, the contractile responses induced by EFS at frequencies of 1–10 Hz in wild-type and M2-receptor KO mice were similar in both pattern and amplitude to those observed in the absence of a NOS inhibitor. By contrast, the preparation isolated from the M3-receptor KO mice began to contract at the same time as EFS (Fig. 2B). Differences in the contractile patterns between wild-type and each M-receptor KO mice were similar to those seen in the absence of L-nitroarginine; the phasic contraction in M3-receptor KO mice was smaller, whereas sustained contraction in M2-receptor KO mice was larger (Fig. 3). Atropine almost completely inhibited the phasic contraction in wild-type and M2- and M3-receptor KO mice (Fig. 4, A–C). In wild-type mice, atropine increased, in a statistically significant manner, the sustained contraction induced by EFS at 10 Hz but not at the lower 1- and 3-Hz levels (Table 1). The sustained contraction observed in wild-type and M3-receptor KO mice was significantly enhanced by atropine, whereas that in M2-receptor KO mice was not (Table 2). Muscle strips prepared from M2- and M3-receptor double-KO mice exhibited a slow contraction in response to EFS of 10 Hz (Fig. 4D). This contraction was larger in amplitude than that observed in wild-type mice and was not affected by atropine. The amplitude of sustained contraction in M4-receptor KO mice was almost equal to that seen in wild-type mice (30.7 ± 4.9% of 60 mM K+-induced contraction; n = 3) (Fig. 4E). Atropine abolished the phasic contraction and increased the sustained contraction in M4-receptor KO mice (Fig. 4E).


Figure 3
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  		Fig. 3. Comparison of EFS-induced contractions in the presence of L-nitroarginine in wild-type and M2- and M3-receptor KO mice. A: phasic contractions at 1, 3, and 10 Hz are expressed as a percentage of 60 mM K+-induced contraction. Note that phasic contractions in M3-receptor KO mice were lower than those of the other 2 types of mice. Values are means ± SE for 4–8 experiments. *Significantly different from values for wild-type mice, P < 0.05. B: sustained contractions at 1, 3, and 10 Hz are expressed as a percentage of 60 mM K+-induced contraction. Note that sustained contractions at 10 Hz in M2-receptor KO mice were higher than those of the other 2 types of mice. Values are means ± SE for 4–8 experiments. *Significantly different from values for wild-type mice, P < 0.05.

 

Figure 4
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  		Fig. 4. Effects of atropine on EFS-induced contractions in the presence of L-nitroarginine. Ileal preparations from wild-type (A) and M2- (B), M3- (C), and M4-receptor KO (E) mice and M2- and M3-receptor double-KO mice (D) were stimulated by EFS at 10 Hz for 10 s. Horizontal lines indicate the presence of 1 µM atropine. After normal spontaneous movements were recorded, the chart was run at a fast speed immediately before the stimulation to make the contraction clear. Bold horizontal lines indicate the duration of EFS for 10 s. Upward arrows indicate the sustained contraction immediately after the termination of EFS.

 

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  		Table 1. Effects of atropine on sustained contraction induced by EFS in the presence of L-nitroarginine

 

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  		Table 2. Comparison of effects of atropine on sustained contraction induced by EFS in the presence of L-nitroarginine between wild-type and M2- and M3-receptor KO mice

 
We next studied the involvement of SP in the mediation of sustained contraction. Treatment of a segment of wild-type mouse ileum with {alpha}-chymotrypsin (3 U/ml) resulted in a decrease in contraction by 23.0 ± 3.0% (n = 5) compared with control (in the absence of drugs) (Fig. 5), suggesting the involvement of peptides. Capsaicin is a sensory neuron stimulant that releases SP and depletes primary sensory afferent neurons (29). To deplete endogenous SP, a segment in the presence of atropine and L-nitroarginine was treated with capsaicin 10–20 min before EFS. Capsaicin inhibited a sustained contraction in a concentration-dependent manner (Fig. 6A). At 100 µM, a sustained contraction was completely inhibited (0.17 ± 0.1% of control) (Fig. 6B). Capsaicin (30 µM) inhibited sustained contractions in M2- and M3-receptor KO mice to an extent similar to that observed in wild-type mice (Fig. 6C). The selective neurokinin NK2-receptor antagonist MEN-10376 (10 µM) significantly inhibited EFS-induced sustained contractions (67.1 ± 5.0% of control) and SP-induced contractions (58.7 ± 4.5% of control) in the ileum of wild-type mice (Fig. 7). By contrast, spantide (3 µM) did not significantly change EFS-induced sustained contractions (100.5 ± 6.8% of control; n = 4).


Figure 5
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  		Fig. 5. Representative trace of effects of {alpha}-chymotrypsin on EFS-induced sustained contraction in the presence of 1 µM atropine and 30 µM L-nitroarginine. The preparations prepared from the ileum of wild-type mice were stimulated by EFS at 10 Hz for 10 s. Horizontal line indicates the presence of {alpha}-chymotrypsin at 3 U/ml. After normal spontaneous movements were recorded, the chart was run at a fast speed immediately before the stimulation to make the contraction clear. Bold horizontal line indicates the duration of EFS for 10 s.

 

Figure 6
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  		Fig. 6. Effects of capsaicin on EFS-induced sustained contraction. A: representative traces of effects of capsaicin on EFS-induced sustained contraction in the presence of 1 µM atropine and 30 µM L-nitroarginine. The preparations prepared from the ileum of wild-type mice were stimulated by EFS at 10 Hz for 10 s. Horizontal line indicates the presence of capsaicin (30 µM and 100 µM). After normal spontaneous movements were recorded, the chart was run at a fast speed immediately before the stimulation to make the contraction clear. Bold horizontal lines indicate the duration of EFS for 10 s. B: summary of effects of capsaicin in wild-type mice. Sustained contractions are expressed as a percentage of 60 mM K+-induced contraction. Values are means ± SE for 4–20 experiments. *Significantly different from values in the absence of capsaicin (control), P < 0.05. C: summary of effects of 30 µM capsaicin in M2- and M3-receptor KO mice. Sustained contractions at 1–10 Hz indicated are expressed as a percentage of 60 mM K+-induced contraction. Values are means ± SE for 3–5 experiments. *Significantly different from values in the absence of capsaicin (control), P < 0.05.

 

Figure 7
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  		Fig. 7. Effects of neurokinin NK2 antagonist MEN-10376 on EFS-induced sustained and substance P (SP)-induced contractions in wild-type mice. A: representative trace of effects of MEN-10376 on EFS-induced sustained contraction in the presence of 1 µM atropine and 30 µM L-nitroarginine. The preparations prepared from the ileum of wild-type mice were stimulated by EFS at 10 Hz for 10 s. Horizontal line indicates the presence of MEN-10376. After normal spontaneous movements were recorded, the chart was run at a fast speed immediately before the stimulation to make the contraction clear. Bold horizontal lines indicate the duration of EFS for 10 s. B: summary of effects of MEN-10376 in wild-type mice. The preparations prepared from the ileum of wild-type mice were stimulated by EFS at 10 Hz for 10 s and SP (100 nM) in the presence of 1 µM atropine and 30 µM L-nitroarginine. Contractions are expressed as a percentage of 60 mM K+-induced contraction. Values are means ± SE for 6 experiments. *Significantly different from the value in the absence of MEN-10376 (control), P < 0.05.

 
ACh induced contraction of wild-type mouse ileum in a concentration-dependent manner (Fig. 8A). A concentration-contraction curve of the ileum prepared from M2-receptor KO mice was almost identical to that of wild-type mice. In the ileum of M3-receptor KO mice, ACh produced a small contraction, compared with those observed in wild-type and M2-receptor KO mice (Fig. 8A). The contraction induced by ACh at 10 µM was about one-half of that seen in wild-type mice. SP induced concentration-dependent contractions in ileal muscle strips of wild-type mice (Fig. 8B). Atropine did not affect the SP-induced contraction (Fig. 8B). The responses to SP did not change in preparations prepared from M2- and M3-receptor KO mice.


Figure 8
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  		Fig. 8. Concentration-contraction curves of ACh (A) and SP (B) in wild-type and M2- and M3-receptor KO mice. A: ACh induced the contraction in a concentration-dependent manner in wild-type ({circ}) and M2- ({triangleup}) and M3-receptor ({square}) KO mice. ACh-induced contractions are expressed as a percentage of 60 mM K+-induced contraction. Note that ACh-induced contractions in the M3-receptor KO mice were lower than those in the other 2 types of mice. Values are means ± SE for 8 experiments. *Significantly different from value for wild-type mice, P < 0.05. B: SP induced the contraction in a concentration-dependent manner in wild-type ({circ}) and M2- ({triangleup}) and M3-receptor ({square}) KO mice. In ileum of wild-type mice, SP-induced contraction was also recorded in the presence of atropine and L-nitroarginine (bullet). SP-induced contractions are expressed as a percentage of 60 mM K+-induced contraction. Values are means ± SE for 3–7 experiments. *Significantly different from values for wild-type mice, P < 0.05.

 
We examined the colocalization of M2 receptors and endogenous SP in wild-type mouse ileum by immunohistochemistry. Immunoreactivity to an SP antibody was localized in the myenteric and submucosal plexus and sparsely in the circular muscle layer. Double staining with synaptophysin, which indicates the location of the enteric plexus, produced a prominent yellow signal (Fig. 9A). Namely, a part of cells positive to anti-synaptophysin antibody was also immunoreactive to anti-SP antibody in the myenteric ganglia (Fig. 9A). As shown in our previous report, a strong immunoreactivity to an anti-M2 receptor antibody was observed in the smooth muscle in both circular and longitudinal layers and the myenteric plexus (31). Double staining with anti-M2 receptor and anti-SP antibodies produced a yellow signal in the myenteric plexus (Fig. 9B). In whole-mount preparations of mouse ileum, SP-immunopositive cells were also stained with anti-M2-receptor antibody in the myenteric plexus layer (Fig. 9B).


Figure 9
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  		Fig. 9. Localization of SP and M2 receptors in the ileum of wild-type mice. A: images of staining with anti-synaptophysin and anti-SP antibodies in the wild-type mouse ileum (top: tissue sections; bottom: myenteric plexus layers in whole-mount preparation). B: images of double staining with anti-M2 receptor and anti-SP antibodies in the wild-type mouse ileum (top: tissue sections; bottom: myenteric plexus layers in whole-mount preparation). LM, longitudinal muscle; CM, circular muscle; MP, myenteric plexus. Scale bars = 50 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the wild-type and M2- and M3-receptor KO mice, the contractile response, which consists of the phasic component (observed immediately after stimulation) and the sustained component (which is persistent after EFS), was observed in the electrically stimulated ileum. The present report confirmed that both contractions were induced through excitation of the enteric neurons by EFS since they were abolished by tetrodotoxin, a nerve-blocking toxin. It is well known that electrical stimulation concurrently activates the inhibitory response in intestinal segments (11). In the present study, the EFS-induced response was changed from contraction to relaxation by treatment with atropine. This relaxation was inhibited by a NOS inhibitor, consistent with our previously report that NO acts in an inhibitory manner in the mouse ileum (30). NO released by EFS may influence the contractile response. Therefore, the present study, except for the experiments described in Figs. 1 and 2, was carried out in the presence of the NOS inhibitor L-nitroarginine.

Involvement of M receptor in EFS-induced phasic contraction. Phasic contractions increased in a frequency-dependent manner in the wild-type and M2- and M3-receptor KO mice and were completely inhibited by atropine. However, the phasic contractions in M3-receptor KO mice was obviously smaller than those observed in wild-type and M2-receptor KO mice. In the absence of L-nitroarginine, the preparation prepared from M3-receptor KO mice did not contract immediately after commencement of electrical stimulation, suggesting an inhibitory effect of NO as a result of a decrease in M-receptor-activated contraction. Also, the responses to exogenous ACh decreased in ileal preparations from M3-receptor KO mice, in conformity with previous reports that the contraction of M3-receptor KO mouse ileum to carbachol was decreased (19). These results indicate that lack of M3 receptor results in a significant decline in ACh-mediated contraction induced by EFS.

In the ileum of M3-receptor KO mice, the EFS-induced phasic contraction was significantly inhibited by atropine, and the exogenous addition of ACh produced a slight contraction. It was reported that the carbachol-induced contraction in M3-receptor KO mice was mediated by the M2 receptor with the rank order of inhibitory effects of antagonists (28, 36). In addition, in the present study, EFS induced only a slow contraction in the ileum of M2- and M3-receptor double-KO mice, and this contraction was unaffected by atropine. Thus the M2 receptor may also be involved in the phasic contraction induced by EFS. However, no clear difference was observed in EFS-induced phasic and ACh-induced contractions between the ileum of wild-type and M2-receptor KO mice. The M2 receptor was suggested to induce contraction that was dependent on voltage-dependent Ca2+ entry (36). Furthermore, it was shown that activation of the M3 receptor leads to the inactivation of voltage-dependent Ca2+ currents (35). Therefore, it seems likely that, in the ileum of wild-type mice, ACh released by EFS induces the phasic contraction through activation of the M3 receptor, which, at the same time, inhibits the M2-receptor pathways.

Involvement of M receptor in EFS-induced sustained contraction. In the ileum prepared from wild-type mice, atropine augmented a sustained contraction induced by EFS at 10 Hz. Because the sustained contraction was not inhibited by atropine and was not influenced by L-nitroarginine, it may be mediated by noncholinergic substances. Thus this is the first report indicating regulation of a noncholinergic contraction via the M receptor. To examine which subtype of M receptors is responsible, sustained contractions in M-receptor KO mice were compared with those in wild-type mice. The sustained contraction in M2-receptor KO mice was larger than that seen in wild-type mice and was unaffected by atropine. On the other hand, a difference was not observed in the amplitude of the sustained contraction of M3- and M4-receptor KO and wild-type mice. The sustained contraction in M3- and M4-receptor KO mice, as well as in the wild-type mice, increased with the addition of atropine. These results suggest that the M2 receptor is involved in the inhibitory modulation of noncholinergic sustained contraction induced by EFS. It was previously shown that, in the ileum of M2- and M3-receptor double KO mice, the noncholinergic contraction elicited by EFS at 10 Hz was larger than that observed in the wild-type mice (18). In the present study, a similar result was obtained, thus supporting our hypothesis that the M2 receptor is critical in sustained contraction.

Identification of substances necessary for sustained contraction. In the present study, EFS-induced sustained contraction, conducted in the presence of atropine and L-nitroarginine, was significantly inhibited by {alpha}-chymotrypsin, capsaicin, and MEN-10376, an antagonist of NK2 receptor (17, 21). This result suggests that SP mediates EFS-induced sustained contraction. First, SP was reported to be degraded by {alpha}-chymotrypsin (22). Second, capsaicin was reported to deplete functional pools of SP (1, 29). Thus it appears that the responses mediated by endogenous SP disappear. Third, NK2 receptors were reported to be present in smooth muscle cells of the ileum of several animal species, including mouse (37), and their activation with SP elicited a contraction of intestinal smooth muscle strips (2–4). The fact that MEN-10376 inhibited sustained contraction implies that SP is involved in EFS-induced sustained contraction through the activation of NK2 receptor.

SP-induced contraction was only partially inhibited by MEN-10376 at a high concentration (10 µM). NK1 receptor was reported to be the primary tachykinin receptor involved in nonadrenergic, noncholinergic contraction in the mouse ileum (24). When we observed the effects of spantide, an inhibitor of NK1 and NK2 receptors, on the sustained contraction, spantide had no effect. Although the reason for an inactivation of spantide is unclear, additional experiments are necessary to confirm whether NK1 receptors are involved in EFS-induced sustained contractions of the mouse ileum.

Treatment of capsaicin also depletes some neurotransmitters other than SP reserved in enteric neurons (1). ATP, calcitonin gene-related peptide, and glutamate are suggested as candidates depleted by capsaicin. Among these, ATP and glutamate induced contractions in gastrointestinal muscle preparations (14, 16). However, an involvement of both substances in EFS-induced sustained contractions could not be apparent in the present study.

It was also reported that prostaglandin was a candidate of endogenous contractile substances, other than ACh, in the gastrointestinal tract (23). We examined the effect of indomethacin, an inhibitor of cyclooxygenase, on EFS-induced contraction in the presence of atropine and L-nitroarginine. Because indomethacin had no effect on EFS-induced sustained contraction (data not shown), prostaglandins are not considered to be mediators of sustained contraction.

Regulation of EFS-induced sustained contraction with M2 receptor. M2 receptors are distributed in smooth muscle cells and enteric neurons in the gastrointestinal tract (13, 31). To clarify the site of action of M2-receptor inhibition of EFS-induced sustained contraction, the responses to SP, added exogenously, were compared in ileal preparations of wild-type and M2- and M3-receptor KO mice. Concentration-contractile response curves for SP in M2-receptor KO mice were similar to those in wild-type and M3-receptor KO mice. In addition, atropine did not affect the SP-induced contraction in wild-type mice. Therefore, it appears that the lack of or the blockade of the M2 receptor has no influence on contraction of ileal smooth muscle strips induced by SP. There were reports that activation of M2 receptors regulated the release of several neurotransmitters (32, 34, 40, 42). Our group (31) also reported that M2 receptors were present on the cholinergic neurons and regulated ACh release from the myenteric plexus. In isolated porcine ileum, atropine was reported to increase the release of SP (26). When we examined the coexpression of M2 receptors and SP in the mouse ileum, an involvement of the M2 receptor in SP release was revealed. Previously, the specificity of anti-M2-receptor antibody has been confirmed in the mouse ileum because the immunoreactivity of this antibody was not observed in M2-receptor KO mice (31). In the present study, localization of M2 receptors on the SP-containing neurons was observed in the myenteric plexus of the mouse ileum. M4 receptors, in addition to M2 receptors, have been reported to play a valuable role in regulating the release of neurotransmitters in both the central and peripheral nervous systems (31, 34, 40). However, M4 receptors had no role to play in the control of EFS-induced sustained contraction as described above. These results suggest the involvement of M2 receptors in regulation of SP release from the enteric neurons.

In conclusion, we have demonstrated that the M3 receptor plays a major role in ACh-mediated phasic contraction in the ileum. By contrast, the M2 receptor has not only a partial role in phasic contraction but also is involved in sustained contraction, probably by negatively modulating the release of SP. It was reported that the lack of M3 receptors had no significant effect on a transit of an intragastrically administrated charcoal in vivo (41). This result suggests that noncholinergic sustained contractions play essential roles in intestinal movement. Therefore, it seems likely that M2 receptors have important functions in the gastrointestinal motility. In addition, decreasing effects of the M2 receptor were not observed in sustained contraction induced by EFS at low frequencies because atropine increased only the sustained contraction induced by EFS at 10 Hz in the ileum of wild-type mice. This result indicates the possibility that M2 receptors control excesses of intestinal motility through the restriction of the excitation of enteric neurons.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by Grants-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science, and by scholarships from Ono Pharmaceutical This study was funded by Pharmacia, Detrol LA Research Grant Program from Pfizer, The Industrial Technology Research Grant Program (02A09001a) from The New Energy and Industrial Technology Development Organization (NEDO), and Grant-in-Aid for Scientific Research (16067101 and 18390073) from The Ministry of Education, Culture, Sports, Science and Technology (MEXT). We thank Toshiya Manabe for encouragement and Shiho Sato and Naoko Numata for technical assistance in mouse breeding.


   ACKNOWLEDGMENTS
 
We thank Dr. Hiroshi Kuramoto (Kyoto Institute of Technology) for helpful suggestions and Dr. G. S. Drummond (InfaCare Pharmaceutical) for the critical reading of this manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Takeuchi, Dept. of Veterinary Pharmacology, Graduate School of Life and Environmental Sciences, Osaka Prefecture Univ., Sakai Osaka 599–8531, Japan (e-mail: takeuchi{at}vet.osakafu-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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